Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5252—Virus inactivated (killed)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/30011—Nodaviridae
  • C12N2770/30034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/30011—Nodaviridae
  • C12N2770/30061—Methods of inactivation or attenuation
  • C12N2770/30063—Methods of inactivation or attenuation by chemical treatment

Definitions

• the present invention relates to vaccines for protecting fish against nodavirus infection, to processes for manufacturing such vaccines, and to use of these vaccines in preventing Viral Nervous Necrosis.
• Beta-nodaviruses are single-stranded RNA, non-enveloped virions and are the etiological agents of Viral Nervous Necrosis (VNN) or fish encephalitis, a disease characterized by the development of a vacuolating encephalopathy and retinopathy, and the presence of viruslike particles in neurons of infected fish.
• VNN Viral Nervous Necrosis
• VNN represents a significant barrier to commercial aquaculture activities because of the frequency of incidence of the disease, the high levels of mortality (approaching 100%) and its widespread distribution across warmwater and coldwater farmed fish species.
• a vaccine against VNN comprising inactivated piscine nodavirus.
• a method of preparing a piscine nodavirus vaccine comprising inactivating the virus using an aziridine compound.
• a third aspect of the invention there is provided a method of treating or preventing VNN comprising administering to a fish in need of such treatment a vaccine comprising inactivated piscine nodavirus.
• inactivated nodavirus in the manufacture of a medicament for the prevention or treatment of nodavirus infection or VNN.
• a strain with similar identifying characteristics is one which serologically reacts with antiserum raised against deposited nodavirus strain Mt/01/Sba.
• a process for the production of a vaccine against a nodavirus starts with infecting suitable culture cells with a virulent strain of the virus in question.
• the method comprises the steps of 1) infecting a susceptible cell line with a piscine nodavirus; 2) allowing said nodavirus to grow in a growth-supporting media until a cytopathic effect (CPE) is produced; 3) harvesting said growth-supporting media containing said nodavirus, dead cells, cell debris and infected cells to produce a harvest material; 4) inactivating said harvest material with a suitable inactivating agent; and 5) adjuvanting said inactivated harvest material.
• CPE cytopathic effect
• Piscine nodaviruses are commonly cultured in Striped snakehead cells (SNN-1), or SBL from sea bass. SSN-1 is available from the ECACC, Salisbury, UK. Another possibility is the Epinephelus coioides grouper cell line disclosed in EP-A-1006178 (GF-1).
• the cells may comprise a mixed population, or they may be cell clones (such as the E-11 cell line described in Iwamoto et al. (2000) Dis. Aquat. Organ. 43(2): 81-9).
• the cell line can be grown in Roux bottles, roller bottles or in bioreactors as a suspension culture or on microcarrier beads using any media and serum that supports rapid growth of the cells.
• the cells are preferably cultivated to form a confluent monolayer and then inoculated with the virus, followed by incubation in a nutrient medium suitable for growth and replication of the virus until the appearance of cytopathic effects (CPE).
• CPE cytopathic effects
• Leibovitz L-15 medium (Gibco) is an example of an appropriate culture medium, as described by Frerichs et al. (1996) J. Gen. Virol. 77:2067-2071.
• the preferred sera for cell growth are fetal equine, fetal bovine or calf serum.
• the culture is clarified and filtered, often with centrifugation, such as CsCl density gradient centrifugation, to remove the cells and cell debris, after which the supernatant fluids may be concentrated by ullrafiltration.
• centrifugation such as CsCl density gradient centrifugation
• the term "inactivated virus” refers to previously virulent virus which has undergone treatment to inactivate, kill or otherwise modify the virus to substantially eliminate its virulent properties while retaining its characteristic property of immunogenicity.
• the virus can be inactivated by addition of an aziridine compound, which may be monomeric (e.g. ethyleneimine) or oligomeric.
• the preferred aziridine compound is binary ethyleneimine (BEI).
• BEI binary ethyleneimine
• Other examples are trimeric ethyleneimine (TEI) and acetyl-ethyleneimine.
• BEI can be prepared by dissolving, to a concentration of 0.1 M, 2-bromoethylamine hydrobromide (BEA) in approximately 0.2 N sodium hydroxide solution, and incubating at 37°C for 1 hour.
• BEA 2-bromoethylamine hydrobromide
• the BEI formed in this way by cyclisation is then added to a virus suspension, the process of inactivation generally requiring a period of at least several hours, preferably at a BEI concentration of from about 0.001 to about 0.01 M, most preferably between about 0.003 to about 0.005M, and optimally about 0.004M.
• the culture is held at normal temperature/room temperature. For instance, an inactivation step in 0.004M BEI at 25°C for 72 hours may be appropriate.
• the pH of the culture medium tends to become more acid over time. Accordingly the pH of the culture medium is optionally monitored continuously during the inactivation process and maintained at the desired mildly alkaline or mildly acidic level by the addition of further alkali as required.
• any residual BEI in the harvest can be neutralised by adding an excess amount of a suitable reagent such as citric acid or sodium thiosulphate solution.
• Ethyleneimine can be prepared by addition of 2-aminoethyl hydrogen sulphate to a boiling sodium hydroxide solution, and is also available commercially. Acid induced polymerization of ethyleneimine produces different oligomeric ethyleneimines; selected oligomers can be isolated from the reaction mixture by fractional distillation. In a manner similar to BEI, ethyleneimine and its oligomer forms are mixed with a viral suspension and incubated in order to effect inactivation.
• the viral inactivation step is carried out at a temperature in the range of about 15 to about 35°C, preferably about 20 to about 27°C, and more preferably about 24- 26°C, optionally at about 25°C.
• the inactivated viral preparation is incubated with susceptible host cells for several days, for instance as described in the Examples, and observed for CPE.
• Preservatives such as thimerosal can be added to the inactivated fluids, and the inactivated material may be adjuvanted. After adjuvanting, stabilizers such as glycerol/EDTA can be added to improve antigen stability.
• the virus fluids may be further concentrated using ultrafiltration, polyethylene glycol precipitation or polyethylene oxide adsorption. These concentrated antigens can be kept at -70°C or lower temperatures for many years, if necessary, and made into vaccine when required by dilution in a suitable buffer and optional addition of adjuvants.
• the inactivated viral supernatant is admixed with a pharmaceutically acceptable carrier, and optionally with an adjuvant.
• the prime targets of the vaccine of the invention are extremely diverse, being any marine or freshwater species which is susceptible to infection by piscine beta-nodaviruses.
• a non- exhaustive list includes: Sea bass (Dicentrarchus labrax L), Sea bream (Sparus aurata), Umbrina (Umbrina cirrosa), Atlantic halibut (Hippoglossus hippoglossus), Winter flounder (Pleuronectes americanus), Atlantic cod (Gadhus morhua), Haddock (Melanogramus aeglefinus), Dover sole (Solea solea), Turbot (Scophthalmus maximus), Jack fish (e.g.
• Grouper e.g. sevenband grouper/ Epinephelus septemfasciatus, redspotted grouper/ Epinephelus akaara, greasy grouper /Epinephelus tauvina, carpet cod /Epinephelus fuscogutatus, humpback g rouper/ Cromileptes altivelis, blackspotted grouper/ Epinephelus malabaricus, kelp grouper/ Epinephelus moara, and marbled leopard grouper/ Plectropomus maculates), Wolffish (Anarhicas minor), Barramundi (Lates calcarifer), tiger puffer (Takifugu rubripes), Japanese flounder (Paralichthys olivaceus), rock porgy (Oplegnathus punctaius) and Japanese Parrotfish (Oplegnaihus fasciatus).
• Grouper e.g. sevenband grouper/ Epinephelus septemfasciatus, redspotted grouper/
• Vaccines prepared according to the invention can comprise any inactivated piscine nodavirus, and can achieve protection against any piscine nodavirus.
• the virus used to prepare the inactivated vaccine of the invention is selected to be the same strain or of the same genotype as the virus against which protection is sought (although there is likely to be good cross-protection between different genotypes).
• Piscine nodaviruses can be divided into multiple genotypes based on partial sequence of the coat protein.
• Non-limiting examples of piscine nodaviruses are: SJNNV (striped jack nervous necrosis virus), RGNNV (redspotted grouper nervous necrosis virus), TPNNV (tiger puffer nervous necrosis virus), BFNNV (barfin flounder nervous necrosis virus), FEV (fish encephalitis virus), MGNNV (malabaricus grouper nervous necrosis virus), DGNNV (dragon grouper nervous necrosis virus), Atlantic halibut nodavirus, sea bass encephalitis virus, Lates calcarifer encephalitis virus, Maltese sea bass nodavirus, and GGNNV (greasy grouper nervous necrosis virus).
• SJNNV striped jack nervous necrosis virus
• RGNNV redspotted grouper nervous necrosis virus
• TPNNV tiger puffer nervous necrosis virus
• BFNNV barfin flounder nervous necrosis virus
• FEV fish encephalitis virus
• MGNNV malabaricus grouper nervous necrosis virus
• nodavirus Strains of nodavirus are available from depositary institutions and laboratories around the world, including the Community Reference Laboratory for Fish Diseases at the Danish Veterinary Institute in Arhus. Examples are GNNV strain MT9410, SJNNV strain SJNag93, RGNNV strain SGWak97, RGNNV strain SGMie95, TPNNV strain TPKag93, BFNNV strain JFIWa98, Halibut nervous necrosis virus isolates AHNor96 and AH99NorA, Umbrina cirrosa nodavirus isolate Uc-1 , Atlantic halibut nodavirus isolate AH95NorA, and Japanese flounder nervous necrosis virus isolate JF-HI93.
• Maltese nodavirus isolate Mt/01/Sba is a preferred strain for use in the present invention. This strain was deposited under the Budapest Treaty at the European Collection of Cell Cultures (ECACC) at Porton Down, Salisbury, Wiltshire SP4 OJG, UK, on June 4, 2003 and assigned Accession Number 03060401. The source of this strain is the Institute of Aquaculture at the University of Stirling. Nodavirus strains with similar identifying characteristics preferred for use in the invention can be identified by specific cross-reaction with antiserum or purified polyclonal or monoclonal antibodies raised against strain Mt/01/Sba.
• ECACC European Collection of Cell Cultures
• the vaccine of the invention provides protection against nodavirus infection and related disease to a degree which is commercially significant and valuable for the fish farming industry. Given that nodavirus infections typically kill nearly 100% of all infected fish in the field, any treatment resulting in an increase in relative percentage survival (RPS) is an improvement over conventional treatment methods. In commercial terms, significant protection usually means an RPS for vaccinated fish of at least about 30%, more preferably at least about 40%, more preferably at least 50%, more preferably at least 60%, for instance at least 70%.
• the vaccine of the invention may be used prophylactically, or may be administered as a treatment to nodavirus infected fish to eliminate the infection and/or improve recovery rates.
• a population of fish of a certain species or of a certain age may not develop any disease symptoms when they are exposed to nodavirus. However, they may act as carriers for the virus, and the risk remains that they may pass the virus to fish which are vulnerable. It is also an aspect of the invention to vaccinate clean brood-stock fish against nodavirus using the inactivated virus disclosed herewith to prevent them from becoming carriers.
• the invention provides use of an inactivated piscine nodavirus in the manufacture of a vaccine for the prevention or treatment of VNN in Atlantic cod, by vaccinating co-cultivated salmonid fish (such as coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha), masu salmon (Oncorhynchus masou), pink salmon (Oncorhynchus gorbuscha), rainbow trout (Oncorhynchus mykiss), and Atlantic salmon (Salmo sala ⁇ ).
• co-cultivated salmonid fish such as coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha), masu salmon (Oncorhynchus masou), pink salmon (Oncorhynchus gorbuscha), rainbow trout (Oncorhynchus mykiss), and Atlantic salmon (Salmo sala ⁇ ).
• co-cultivated salmonid fish such as coh
• the vaccine of the invention is administered to larvae and/or juveniles of a fish species, such as juveniles of sea bass.
• the vaccine can be administered to adult or mature fish.
• the typical routes of administration of the vaccine are by injection into the peritoneal cavity (for larger fish), intramuscular injection, orally in feed, or by immersion in seawater or in fresh water. It is recommended that fish be 5 grams or greater in body weight for administration of the vaccine of the invention by injection.
• a suitable injection volume is about 10 to about 200 ⁇ l, preferably about 50 to about 100 ⁇ l.
• a body weight of al least 1 gram is preferred.
• the effective dosage of vaccine may vary depending on the size and species of the subject, and according to the mode of administration.
• the optimal dosage can be determined through trial and error by a veterinarian or aquaculture specialist.
• a suitable dosage range of virus is from about 10 2 to 10 9 TCID 50 (tissue culture infectious dose affecting 50% of cultures inoculated) per unit dose, preferably about 10 4 to 10 8 TCID 50 per unit dose, more preferably about 10 6 to 10 7 TCID 50 per unit dose, most preferably about 10 7 TCID 50 per unit dose.
• TCID 50 tissue culture infectious dose affecting 50% of cultures inoculated
• Preferably a single dosage unit is administered to the fish to be treated. Smaller fish may benefit from a dose of about 10 4 to 10 7 TCID 50 /ml with dip (immersion) administration, for instance with a contact time of about 60 seconds.
• vaccines are prepared as liquid solutions, emulsions or suspensions for injection or delivery in water.
• a liquid emulsion or emulsifiable concentrate can be prepared in order to be added to a water tank or bath where the fish are held.
• Solid (e.g. powder) forms suitable for dissolution in, or suspension in, liquid vehicles, or for mixing with solid food, prior to administration may also be prepared.
• the vaccine may be a lyophilised culture in a ready to use form for reconstitution with a sterile diluent.
• lyophilized cells may be reconstituted in 0.9% saline (optionally provided as part of the packaged vaccine product).
• a preferred formulation of injectable vaccine is an emulsion.
• Liquid or reconstituted forms of the vaccine may be diluted in a small volume of water (e.g. 1 to 100 volumes) before addition to a pen, tank or bath.
• the pharmaceutical vaccine compositions of the invention may be administered in a form for immediate release or extended release.
• Pharmaceutically acceptable carriers or vehicles with which the inactivated virus can be admixed include conventional excipients, and may be, for example, aqueous solvents such as water, saline or PBS, oil, dextrose, glycerol, wetting or emulsifying agents, bulking agents, stabilizers, anti-oxidants, coatings, binders, fillers, disintegrants, diluents, lubricants, pH buffering agents, and the like.
• aqueous solvents such as water, saline or PBS, oil, dextrose, glycerol, wetting or emulsifying agents, bulking agents, stabilizers, anti-oxidants, coatings, binders, fillers, disintegrants, diluents, lubricants, pH buffering agents, and the like.
• Adjuvants such as muramyl dipeplides, avridine, aluminium hydroxide, aluminium phosphate, oils, oil emulsions, saponins, dextran sulphate, glucans, cytokines, block co-polymers, immunostimulatory oligonucleotides and others known in the art may be admixed with the inactivated viral supernatant.
• a preferred adjuvant is Freund's Incomplete Adjuvant (FIA).
• the amount of adjuvant added depends on the nature of the adjuvant itself.
• FIA may advantageously be emulsified with inactivated viral supernatant in a ratio of about 1:1 by volume.
• antigens with which the inactivated nodavirus may be combined include antigens from: Iridovirus, VHSV, Photobactehum damselae subsp. piscicida, Aeromonas spp., Vibrio spp., Edwardsiella spp., Lactococcus spp., Streptococcus spp., Flexibacter spp. and Nocardia spp.
• SSN-1 cells are grown in L15 medium (Invitrogen) supplemented with 10% foetal bovine serum (FBS) at 28°C. To subculture these cells, confluent monolayers (4-7 days old) are washed twice using PBS Dulbeccos (Invitrogen) and harvested using trypsin-EDTA (Invitrogen). One flask of cells is routinely split to produce 3 daughter flasks (split ratio 1:3).
• a confluent 75cm 2 flask of SSN-1 cells is subcultured to produce a 175cm 2 flask and a 25cm 2 flask. These are incubated at 28°C until the cells are 70-80% confluent (usually 1 day old).
• the conditioned media is then aseptically removed and retained and 5ml of Hanks Balanced Salt Solution (HBSS ; Invitrogen) plus 3mi nodavirus from Maltese sea bass fry (Mt/01/Sba) are added to the 175cm 2 flask.
• HBSS Hanks Balanced Salt Solution
• Mt/01/Sba Maltese sea bass fry
• the 25cm 2 flask is used as a mock (negative control) and has 1 ml conditioned media and 1 ml HBSS + 2% FBS only added to it. Flasks are incubated at 25°C for 30 minutes and then resupplemented as follows:
• TClD 50 tissue culture infectious dose affecting 50% of cultures inoculated
• TClD 50 tissue culture infectious dose affecting 50% of cultures inoculated
• BEI back titration
• Samples (a) - (c) act as negative controls and demonstrate the effect these chemicals might have on the cells.
• Samples (d) and (e) are the positive controls and allow the TCID 50 to be calculated using the Spearman-Karber method.
• Sample (e) also shows whether the sodium thiosulphate alone has an effect on the virus.
• Samples (f)-(h) are replicates of the inactivated virus and allow to check that the virus is no longer viable.
• the 96-well plate is incubated at 25°C and monitored daily. A final reading is taken after 7- 10 days incubation and the TCID 50 calculated.
• the above samples are also simultaneously inoculated into 25cm 2 flasks of SSN-1 cells in L15 media and 5% FBS and incubated at 25°C. After 7 days incubation these cell cultures are passaged onto 1 day-old SSN-1 cells (pre-formed monolayers). A further two successive pre-formed passages are carried out using day 7 supernatant from the previous passaged flask as the inoculum. All of the flasks are monitored for a total of 21 days to check for CPE. All flasks containing BEI treated virus remain negative for CPE (all other controls are as expected); thus, it can be assumed that the virus has been successfully inactivated. Inactivated virus is emulsified 1:1 in Freund's Incomplete Adjuvant (FIA), to a concentration of 10 8 TClD 50 /ml.
• FFA Freund's Incomplete Adjuvant
• Recombinant antigen is prepared by cloning the complete coding sequence of the capsid protein from halibut nodavirus strain V9954 into the pET-30EK/LIV vector (Novagen). Following induction of the host cell culture with IPTG, the recombinant capsid protein is harvested as a non-soluble product (inclusion bodies). The recombinant protein is subsequently solubilized, then refolded by performing several rounds of dialysis. The final recombinant antigen is emulsified 1:1 in Freund's Incomplete Adjuvant (FIA), to a final protein concentration of 0.675 mg/ml.
• FIA Freund's Incomplete Adjuvant
• Emulsified vaccines and controls are made by dropping (at about 1 drop sec "1 ) FIA into sterile 15ml Falcon tubes containing the respective vaccine or control, while vortexing.
• Sea bass (Dicentrarchus labrax) of mean weight 18g are kept in tanks of approximately 40L each, with 60 or 65 fish to a tank.
• the tanks are supplied with recirculating UV and ozone (when necessary) treated salt-water (35%o) system at 23+1° C, and fed commercial sea bass feed (SOR ⁇ AL S.A., Ovar, Portugal).
• fish After acclimatizing for 7 days in the new holding system and being starved for 24 hours, fish are anaesthetized and immunized with one of the following treatments in duplicated groups by intraperitoneal injection (100 ⁇ l/fish): recombinant antigen 1:1 in FIA; BEI inactivated virus 1:1 in FIA, PBS 1 :1 in FIA (control); HBSS (Hank's Balanced Salt Solution/Gibco BRL) 1:1 in FIA (control).
• recombinant antigen 1:1 in FIA BEI inactivated virus 1:1 in FIA, PBS 1 :1 in FIA (control); HBSS (Hank's Balanced Salt Solution/Gibco BRL) 1:1 in FIA (control).
• Virulent nodavirus inoculum is obtained from a Maltese nodavirus isolate (Mt/01/Sba) originating from farmed sea bass juveniles. Tissue cultures of original harvest are simultaneously inoculated into SSN-1 cells using L-15 medium supplemented with 5% FBS and incubated at 25°C. When CPE is complete, tissue cultures are clarified by centrifugation at 1500g for 15 min and the supernatant containing virus is re-inoculated (preformed inoculation) into SSN-1 lines in order to achieve a high concentration of virus.
• Mt/01/Sba Maltese nodavirus isolate
• tissue cultures are centrifuged (1500g for 15 min) and stocked at -70°C until use. SSN-1 cells are then added and incubated with the virus at 25°C.
• the TCID 50 tissue culture infectious dose infecting 50% of inoculated cultures is calculated according to the method of Spearman-Karber (1931).
• the challenge inoculum is diluted in HBSS (Gibco) supplemented with 2% FBS to obtain a challenge dose of 10 7 TCID 50 per fish.
• HBSS Gibco
• FBS 2% FBS
• each fish is inoculated i.p. with 100 ⁇ l inoculum.
• the temperature of the tank water is increased to 26°C 3 days after the challenge to mimic a natural viral outbreak. Brain tissue samples are taken from dead fish to confirm presence of virus.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Virology (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Biomedical Technology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Farming Of Fish And Shellfish (AREA)

Priority Applications (12)

Applications Claiming Priority (4)

Related Child Applications (2)

Publications (1)

Family

ID=32910468

Family Applications (1)

Country Status (18)

Cited By (3)

Families Citing this family (3)

Citations (5)

Family Cites Families (2)

• 2004
  • 2004-02-17 TW TW093103736A patent/TWI347364B/zh not_active IP Right Cessation
  • 2004-02-18 MY MYPI20040527A patent/MY143785A/en unknown
  • 2004-02-18 DK DK04712006T patent/DK1597359T3/da active
  • 2004-02-18 CL CL200400282A patent/CL2004000282A1/es unknown
  • 2004-02-18 PT PT04712006T patent/PT1597359E/pt unknown
  • 2004-02-18 US US10/545,528 patent/US20060257425A1/en not_active Abandoned
  • 2004-02-18 EP EP04712006A patent/EP1597359B1/de not_active Expired - Lifetime
  • 2004-02-18 ES ES04712006T patent/ES2328141T3/es not_active Expired - Lifetime
  • 2004-02-18 DE DE602004022139T patent/DE602004022139D1/de not_active Expired - Lifetime
  • 2004-02-18 SI SI200431251T patent/SI1597359T1/sl unknown
  • 2004-02-18 CA CA2512930A patent/CA2512930C/en not_active Expired - Lifetime
  • 2004-02-18 JP JP2006501877A patent/JP4664901B2/ja not_active Expired - Lifetime
  • 2004-02-18 AT AT04712006T patent/ATE437224T1/de not_active IP Right Cessation
  • 2004-02-18 AU AU2004213566A patent/AU2004213566B2/en not_active Expired
  • 2004-02-18 WO PCT/EP2004/001543 patent/WO2004074467A1/en active Application Filing
• 2005
  • 2005-09-12 NO NO20054219A patent/NO336878B1/no unknown
• 2006
  • 2006-05-17 HK HK06105723.7A patent/HK1085765A1/xx not_active IP Right Cessation
• 2009
  • 2009-04-16 US US12/425,135 patent/US8124100B2/en not_active Expired - Lifetime
  • 2009-08-28 CY CY20091100898T patent/CY1109344T1/el unknown

Patent Citations (6)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events